Compositions and methods for c. difficile treatment

ABSTRACT

The present disclosure provides compositions and methods for treating  Clostridium difficile  infection (CDI) including primary and recurrent CDI. In particular, the compositions and methods described herein are capable of achieving a CDI clearance rate of at least 80% through a single oral dose of pharmaceutical composition comprising a freeze-dried fecal microbiota preparation.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/357,814, filed Jul. 1, 2016, and U.S. Non-Provisional applicationSer. No. 15/258,821, filed Sep. 7, 2016, which are herein incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present disclosure generally relates to medicine andgastroenterology, pharmacology, and microbiology. In particular, thisapplication provides methods for treating Clostridium infection (CDI)that cannot be completely cleared with antibiotics alone.

BACKGROUND OF THE INVENTION

Widespread usage of antimicrobial drugs over many decades in medicineand agriculture has resulted in emergence of increasing numbers ofantibiotic-resistant pathogens, which constitute one of the most urgentgrowing threats in modern healthcare. In addition, antibiotics increasevulnerability to infections by lowering colonization resistance that isnormally provided by the host's own microbiota. Therefore, more targetedtreatments against pathogens that can spare the host microbiota and/orrestorative treatments that can recover the normal host microbiotacomposition is desired.

The syndrome of recurrent Clostridium difficile infection (R-CDI) is acommon clinical challenge that captures the essential pitfalls ofreliance on broad-spectrum antibiotics for treatment. The standardantibiotics for this infection, e.g., metronidazole and vancomycin,suppress gut microbiota leading to a disruption of normal microbialcommunity structure and a decrease in overall microbial diversity. CDIreoccurs after cessation of antibiotic therapy, when the loss ofprotective microbiota and lack of secondary bile acids allow C.difficile spore germination, expansion of vegetative forms of C.difficile bacteria and production of endotoxins.

Recurrent CDI is one of the most difficult and increasingly commonchallenges associated with CDI (Surawicz, Gastroenterology 2009;136:1152-4). An initial incidence of CDI can be followed by a relapsewithin 30 days in about 20-30% of cases (Kelly and LaMont. N Engl J Med2008; 359:1932-40, Louie et al. N Engl J Med 2011; 364:422-31, Pepin etal. Clin Infect Dis 2006; 42:758-64), and the risk of recurrence doublesafter two or more occurrences (McDonald et al. Emerg Infect Dis 2006;12:40915). Older age, intercurrent antibiotic use for non-C. difficileindications, renal insufficiency, immune deficiency, and antacidmedications, are some of the known risk factors for recurrent CD(Surawicz, Gastroenterology 2009; 136:1152-4, Garey et al. J Hosp Infect2008; 70:298-304). The presence of three clinical criteria: age>65years, severe disease, and continued use of antibiotics after treatingthe initial CDI episode, are predictive of an almost 90% relapse rate(Hu et al. Gastroenterology 2009; 136:1206-14). CDI also commonlycomplicates management of inflammatory bowel disease (IBD), which hasrecently been recognized as an additional independent risk factor forCDI infection (Issa et al. Clin Gastroenterol Hepatol 2007; 5:345-51,Rodemann et al. Clin Gastroenterol Hepatol 2007; 5:339-4415). CDI inpatients with underlying IBD is associated with increased severity ofcolitis and higher rates of recurrence and colectomy (Issa et al.Inflamm Bowel Dis 2008; 14:1432-42).

It has been suggested that the presence of normal, healthy, intestinalmicrobiota (normal gut microorganisms) offers protection against CDI.Conversely, severe disruption of normal intestinal microbiota by use ofantibiotics, including metronidazole and vancomycin that are used totreat CDI, is likely one of the major reason for its recurrence. Changand colleagues used 16S rDNA sequencing to analyze the fecal microbiotaof seven patients with initial and recurrent CDT (Chang et al. J InfectDis 2008; 197:435-8). Their report asserts that bacterial speciesdiversity was reduced in all patients compared to nominal controlsubjects. Similarly, Khorats and colleagues reported marked dysbiosis inpatients with CDI as compared to controls using TRFLP analyses of fecalmicrobiota (Khoruts et al. J Clin Gastroenterol. 2010; 44:354-60). Thegreatest reduction in species diversity, however, was found in the threepatients with recurrent CDI and disruption of their gut microbiota wasevident at the phylum level—with marked reduction in Bacteriodetes,normally one of the two dominant phyla in the colon. Instead, the gutmicrobiota in these patients were dominated by members of theProteobacteria and Verrucomicrobia phyla, which usually are only minorconstituents of the colon microbiota.

Fecal microbiota transplantation (FMT), also known as ‘fecalbacteriotherapy,’ represents the one therapeutic protocol that allowsthe fastest reconstitution of a normal composition and functional gutmicrobial community. For many decades, FMT has been offered by selectcenters across the world, typically as an option of last resort forpatients with recurrent Clostridium difficile infection (CDI). Acommonly cited early report for FMT was by Eiseman and colleagues who in1958 described the use of fecal enemas for patients who likely hadsevere or fulminant form of pseudomembranous colitis (Eiseman et al.Surgery 1958; 44:854-9). Since this time, well over 500 cases have beenreported as individual case reports, small case series, or clinicaltrials with a ˜90% cumulative success rate in clearing recurrent CDI,without any noted adverse events. The history and general methodologyused for FMT have been described in several recent reviews (Bakken.Anaerobe 2009; 15:285-9, van Nood et al. Euro Surveill 2009; 14, Khorutsand Sadowsky. Mucosal Immunol 2011; 4:4-7, Khoruts and Sadowsky Nat RevGastroenterol Hepatol. 2016:doi: 10.1038/nrgastro.2016.98).

A recent randomized, controlled clinical study has confirmed theremarkable efficacy of this therapeutic approach (van Nood et al., 2013,N Engl J Med, 368:407-15). However, despite the long and successfultrack record, as well as great clinical need, the availability of theprocedure for many patients remains very limited.

Currently, FMT is administered by several routes including infusion ofhuman microbiota in the form of homogenized stool, extracts ofhomogenized stool, or cultured stool components through a colonoscope,an enema, or via a nasojejunal tube. Although Youngster et al. JAMA 2014asserts that encapsulated, frozen microbiota can be delivered orally andresults in successful treatment of R-CDI, the practicality of thispreparation is limited by esthetic, storage, and shelf-life issues.Against this backdrop, the present disclosure provides a next-generationform of capsule FMT using a freeze-dried preparation of microbiota thatcould tolerate a range of temperatures to allow ease of handling,administration, and storage. The methods and compositions described heresatisfy several conditions: (1) the freeze-drying procedure preserve theviability of the majority of the entire taxonomic spectrum ofmicrobiota, (2) the resulting material have physicochemical propertiesthat enable standardized encapsulation, (3) the encapsulation proceduredoes not compromise the viability of microbiota, and (4) the microbiotaengrafts into the colon and successfully treat R-CDI. Furthermore, theinstant disclosure achieves high CDI clearance rate in a single dose.

SUMMARY OF THE INVENTION

The present disclosure comprises methods for treating Clostridiumdifficile infections (CDI) in subjects in need thereof. In some aspects,the present disclosure includes methods for treating a primary CDI. Inother aspects, the present disclosure includes methods for treating arecurrent CDI that cannot be cleared with antibiotics alone.

More particularly, in an aspect, the method of the present disclosurecomprises orally administrating to a subject in need thereof a singledose of a pharmaceutical composition comprising a freeze-dried fecalmicrobrobe preparation, where the single dose is capable of achieving aCDT clearance rate of at least 80% in a population of the subjectsreceiving the single dose of the pharmaceutical composition.

A further aspect of the present disclosure is that the method of thepresent disclosure comprises orally administrating to a subject in needthereof a single dose of a pharmaceutical composition comprising afreeze-dried fecal microbe preparation, where the single dose is capableof achieving at least 80% CDI clearance rate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a distribution of phyla among all mouse fecal pellets anddonor samples, without rarefication in accordance with Example 2 of thepresent disclosure.

FIG. 1B shows the Phylum-level classification of OTUs that wereassociated with donor contribution in accordance with Example 2 of thepresent disclosure

FIG. 2 shows a principal coordinate analysis of donor and germ-freemouse samples gavaged with PBS control, frozen, or freeze-dried fecalmicrobiota in accordance with Example 2 of the present disclosure.

FIG. 3A shows the distribution of phyla in cured patient and donorsamples in accordance with Example 5 of the present disclosure.

FIG. 3B shows the phylum-level classification of OTUs that wereassociated with donor contribution in accordance with Example 5 of thepresent disclosure.

FIG. 4 shows the alpha diversity within the Bacteroidetes and Firmicutesphyla, individually, in patients pre- and post-FMT cured by FMT, and thedonor samples in accordance with Example 5 of the present disclosure.

FIG. 5 shows the distribution of phyla and similarity to donor (i.e.,attribution of DNA sequences to donor engraftment, as determined byusing the SourceTracker software package) among samples from patientsadministered low (2.1-2.5×10¹¹ cells) and high (1.25-2.5×10¹² cells)doses of capsule FMT in accordance with Example 5 of the presentdisclosure.

FIG. 6 shows the distribution of phyla and total donor similarity amongsamples from patients grouped by use of PPI in accordance with Example 5of the present disclosure.

FIG. 7A shows the distribution of phyla and alpha diversity amongpatients who experienced recurrence of C. difficile infection followinginitial capsule FMT in accordance with Example 5 of the presentdisclosure.

FIG. 7B shows the distribution of phyla and alpha diversity amongpatients who experienced recurrence of C. difficile infection followingcapsule FMT following recurrence after colonoscopic FMT in accordancewith Example 5 of the present disclosure.

Corresponding reference characters indicate corresponding partsthroughout the several views. The example(s) set out hereinillustrate(s) several aspects of the present disclosure but should notbe construed as limiting the scope of the present disclosure in anymanner.

DETAILED DESCRIPTION

This description is not intended to be a detailed catalog of all thedifferent ways in which the disclosure may be implemented, or all thefeatures that may be added to the instant disclosure. For example,features illustrated with respect to one aspect may be incorporated intoother aspects, and features illustrated with respect to a particularaspect may be deleted from that aspect. Thus, the disclosurecontemplates that in some aspects of the disclosure, any feature orcombination of features set forth herein can be excluded or omitted. Inaddition, numerous variations and additions to the various aspectssuggested herein will be apparent to those skilled in the art in lightof the instant disclosure, which do not depart from the instantdisclosure. In other instances, well-known structures, interfaces, andprocesses have not been shown in detail in order not to unnecessarilyobscure the invention. It is intended that no part of this specificationbe construed to effect a disavowal of any part of the full scope of theinvention. Hence, the following descriptions are intended to illustratesome particular aspects of the disclosure, and not to exhaustivelyspecify all permutations, combinations and variations thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. The terminology used in thedescription of the disclosure herein is for the purpose of describingparticular aspects only and is not intended to be limiting of thedisclosure.

All publications, patent applications, patents and other referencescited herein are incorporated by reference in their entireties.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the disclosure described herein can be used inany combination. Moreover, the present disclosure also contemplates thatin some aspects of the disclosure, any feature or combination offeatures set forth herein can be excluded or omitted.

Methods disclosed herein can comprise one or more steps or actions forachieving the described method. The method steps and/or actions may beinterchanged with one another without departing from the scope of thepresent invention. In other words, unless a specific order of steps oractions is required for proper operation of the aspect, the order and/oruse of specific steps and/or actions may be modified without departingfrom the scope of the present invention. For example, the steps may beconducted in any feasible order. And, as appropriate, any combination oftwo or more steps may be conducted simultaneously.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

The terms “about,” “approximately,” and “substantially” as used hereinwhen referring to a measurable value such as a percentage, cell count,volume and the like, is meant to encompass variations of ±20%, ±10%,±5%,±1%, 0.5%, or even ±0.1% of the specified amount.

As used herein, phrases such as “between X and Y” and “between about Xand Y” should be interpreted to include X and Y. As used herein, phrasessuch as “between about X and Y” mean “between about X and about Y” andphrases such as “from about X to Y” mean “from about X to about Y.”

As used herein, “CDI clearance” refers to a lack of spontaneous relapseof diarrheal symptoms and absence of C. difficile toxin B in stoolswithin two months of administrating a therapeutic agent.

As used herein, “absence of C. difficile toxin B” refers to the absenceof detectable C. difficile toxin B DNA tested by PCR. See e.g. Petersonet al Clin. Infect. Dis. 2007; 45:1152-60.

As used herein, “lyophilization” or “freeze drying” refers to theprocess of drying a material by first freezing it and then encouragingthe ice within it to sublimate in a vacuum environment.

As used herein, a “cryoprotectant” refers to a substance that is addedto a formulation in order to protect an active ingredient duringfreezing, e.g. microbial cells.

As used herein, a “lyoprotectant” refers to a substance that is added toa formulation in order to protect an active ingredient during the dryingstage of a Lyophilization (also known as freeze-drying) process.

As used herein, the term “ambient temperature” refers to the temperatureof the surrounding environment, and more specifically, the temperatureof the surrounding air. The term “room temperature” refers to the indoortemperature of a temperature-controlled building, which is approximatelybetween 15° C. (59° F.) and 22° C. (72° F.).

As used herein, “fecal bacteria” refers to bacteria that can be found infecal matter.

As used herein, “fecal microbe” refers to one or more microbes that canbe found in fecal matter.

As used herein, a “microbiota” and “flora” refer to a community ofmicrobes that live in or on a subject's body, both sustainably andtransiently, including eukaryotes, archaea, bacteria, and viruses(including bacterial viruses (i.e., phage)). A “fecal microbiota” or“fecal microbiota preparation” refers to a community of microbes presentin a subject's feces. A non-selected fecal microbiota refers to acommunity or mixture of fecal microbes derived from a donor's fecalsample without selection and substantially resembling microbialconstituents and population structure found in such fecal sample.

As used herein, the term “non-floral fecal material” refers tocomponents of feces that are not microbial in nature. For example,non-floral fecal material includes, without limitation, undigested fiberor host cell debris.

As used herein, “viable” means possessing an intact cell membrane. Here,the viability of bacterial populations is monitored as a function of themembrane integrity of the cell. Cells with a compromised membrane areconsidered to be dead or dying, whereas cells with an intact membraneare considered live. For example, SYTO 9 and propidium iodide are usedto stain and differentiate live and dead bacteria. See Stocks, CytomeryA. 2004 October; 61(2):189-95. Cell viability can also be evaluated viamolecular viability analyses, e.g., a PCR-based approach, which candifferentiate nucleic acids associated with viable cells from thoseassociated with inactivated cells. See Cangelosi and Mescheke, ApplEnviron Microbiol. 2014 October; 80(19): 5884-5891.

As used herein, “isolated” or “purified” refers to a bacterium or otherentity or substance that has been (1) separated from at least some ofthe components with which it was associated when initially produced(whether in nature or in an experimental setting), and/or (2) produced,prepared, purified, and/or manufactured by the hand of man. Isolated orpurified bacteria can be separated from at least about 10%, about 20%,about 30%, about 40%0, about 50%, about 60%, about 70%, about 800, about90%, or more of the other components with which they were initiallyassociated.

As used herein, the terms “pathogen” and “pathogenic” in reference to abacterium or any other organism or entity includes any such organism orentity that is capable of causing or affecting a disease, disorder orcondition of a host organism containing the organism or entity.

As used herein, “spore” or a population of “spores” includes bacteria(or other single-celled organisms) that are generally viable, moreresistant to environmental influences such as heat and bacteriocidalagents than vegetative forms of the same bacteria, and typically capableof germination and out-growth. “Spore-formers” or bacteria “capable offorming spores” are those bacteria containing the genes and othernecessary abilities to produce spores under suitable environmentalconditions.

As used herein, “subject” refers to any animal subject including humans,laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows,sheep, goats, pigs, turkeys, chickens), and household pets (e.g., dogs,cats, rodents, etc.). The subject or patient may be healthy, or may besuffering from an infection due to a gastrointestinal pathogen or may beat risk of developing or transmitting to others an infection due to agastrointestinal pathogen.

As used herein, “Shannon Diversity Index” refers to a diversity indexthat accounts for abundance and evenness of species present in a givencommunity using the formula H=−Σ_(i=1) ^(R) p_(i) ln p_(i), where H isShannon Diversity Index, R is the total number of species in thecommunity, and p_(i) is the proportion of R made up of the ith species.Higher values indicate diverse and equally distributed communities, anda value of 0 indicates only one species is present in a given community.For further reference, see Shannon and Weaver, (1949) The mathematicaltheory of communication. The University of Illinois Press, Urbana.117pp.

As used herein, “antibiotic” refers to a substance that is used to treatand/or prevent bacterial infection by killing bacteria, inhibiting thegrowth of bacteria, or reducing the viability of bacteria.

As used herein, “treatment” or “treating,” with respect to a conditionor a disease, is an approach for obtaining beneficial or desired resultsincluding preferably clinical results after a condition or a diseasemanifests in a patient. Beneficial or desired results with respect to adisease include, but are not limited to, one or more of the following:improving a condition associated with a disease, curing a disease,lessening severity of a disease, delaying progression of a disease,alleviating one or more symptoms associated with a disease, increasingthe quality of life of one suffering from a disease, prolongingsurvival, and any combination thereof. Likewise, for purposes of thisdisclosure, beneficial or desired results with respect to a conditioninclude, but are not limited to, one or more of the following: improvinga condition, curing a condition, lessening severity of a condition,delaying progression of a condition, alleviating one or more symptomsassociated with a condition, increasing the quality of life of onesuffering from a condition, prolonging survival, and any combinationthereof.

As used herein, “prevention” or “preventing,” with respect to acondition or a disease, is an approach for reducing the risk ofdeveloping a condition or a disease before it manifests in a patient.Prevention approaches include, but are not limited to: identifying adisease at its earliest stage so that prompt and appropriate managementcan be initiated, protecting a tissue prone to a condition or a diseaseprior to its manifestation, reducing or minimizing the consequences of adisease, and a combination thereof.

As used herein, “therapeutically effective amount” or “pharmaceuticallyactive dose” refers to an amount of a composition which is effective intreating the named disease, disorder or condition.

As used herein, “a single dose of a pharmaceutical composition” refersto providing a therapeutically effective amount of a composition in asingle administration.

As used herein, “alpha diversity” refers to the mean species diversityat a local scale or a specific habitat and is determined by the numberof species.

As used herein, “dysbiosis” refers to a microbial imbalance ormaladaptation inside the digestive tract.

An aspect of the disclosure includes a method for treating a CDI in asubject in need thereof. In some aspects, a method for treating aprimary CDI in a subject in need thereof is provided. In certainaspects, a method for treating a recurrent CDI in a subject in needthereof is provided. In another aspect, this disclosure provides amethod for preventing a CDT in a subject in need thereof.

In an aspect, a method of the present disclosure comprises orallyadministrating to a subject in need thereof a single dose of apharmaceutical composition comprising a freeze-dried fecal microbepreparation, where the single dose is capable of achieving a CDTclearance rate of at least 80% in a population of the subjects receivingthe single dose of the pharmaceutical composition. In another aspect, asingle dose of the pharmaceutical composition may be capable ofachieving a CDI clearance rate of at least 60% in a population of thesubjects receiving the single dose of the pharmaceutical composition. Incertain aspects, a single dose of the pharmaceutical composition may becapable of achieving a CDI clearance rate of at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, or at least 75% in apopulation of the subjects receiving the single dose of thepharmaceutical composition. In certain aspects, a single dose of thepharmaceutical composition may be capable of achieving a CDI clearancerate of at least 85%, at least 90%, at least 92%, at least 94%, at least96%, at least 98%, or at least 99% in a population of the subjectsreceiving the single dose of the pharmaceutical composition. In anotheraspect, a single dose of the pharmaceutical composition may be capableof achieving a CDI clearance rate of between 50 and 55%, between 55 and60%, between 60 and 65%, between 65 and 70%, between 70 and 75%, between75 and 80%, between 80 and 85%, between 85 and 90%, between 90 and 95%,between 95 and 100%, in a population of the subjects receiving thesingle dose of the pharmaceutical composition. In an aspect, within thefirst two weeks from orally administrating a single dose of thepharmaceutical composition of the present application, the subjectexperience little or no bowel movement irregularity, bloating, orflatulence.

In one aspect, a fecal microbe preparation described herein comprises apurified or reconstituted fecal bacterial mixture. In one aspect, afecal microbe preparation described herein comprises a fecal microbiotapreparation. In one aspect, a fecal microbe preparation comprises one ormore, one or more, two or more, three or more, four or more, or five ormore live fecal microorganisms are selected from the group consisting ofAcidaminococcus, Akkermansia, Alistipes, Anaerotruncus, Bacteroides,Bifidobacterium, Blautia, Butyrivibrio, Clostridium, Collinsella,Coprocxcus, Corynebacterium, Dorea, Enterococcus, Escherichia,Eubacterium, Faecalibacterium, Haemophilus, Holdemania, Lactobacillus,Moraxella, Parabacteroides, Prevotella. Propionibacterium, Raoultella.Roseburia, Rummtococcus, Staphylococcus, Streptococcus, Subdoligranulum,and Veillonella. In one aspect, a fecal microbe preparation comprisesone or more, one or more, two or more, three or more, four or more, orfive or more live fecal microorganisms are selected from the groupconsisting of Bacteroides fragilis ssp. vulgatus, Collinsellaaerofaciens, Bacteroides fragilis ssp. thetaiotaomicron,Peptostreptococcus productus II, Parabacteroides distasonis,Faecalibacterium prautsnitzii, Coprococcus eutactus, Pepostreptococcusproductus, Ruminococcus bromii, Bifidobacterium adolescentis, Gemmigerformicilis, Bifidobacterium longum, Eubacterium siraeum, Ruminococcustorques, Eubacterium rectale, Eubacterium eligens, Bacteroideseggerthii, Clostridium leptum, Bacteroides fragilis ssp. A, Eubacteriumbiforne, Bifidobacterium infantis, Eubacterium rectale, Coprococcuscomes, Pseudoflavonifractor capillosus, Ruminococcus albus, Doreaformicigenerans, Eubacterium hallii, Eubacterium ventriosum,Fusobacterium russi, Ruminococcus obeum, Eubacterium rectale,Clostridium ramosum, Lactobacillus leichmannii, Ruminococcus callidus,Butyrivibrio crossotus, Acidaminococcus fermentans, Eubacteriumventriosum, Bacteroides fragilis ssp. fragilis, Coprococcus catus,Aerostipes hadrus, Eubacterium cylindroides, Eubacterium ruminantium,Staphylococcus epidermidis, Eubacterium limosum, Tissirella praeacuta,Fusobacterium mortiferum, Fusobacterium naviforme, Clostridium innocuum,Clostridium ramosum, Propionibacerium acnes, Ruminooccus flavefaciens,Bacteroides fragilis ssp. ovatus, Fusobacterium nucleatum, Fusobacteriummortiferum, Escherichia coli, Gemella morbillorum, Finegoldia magnus,Streptococcus intermedius, Ruminooccus lactaris, Eubacterium tenue,Eubaclerium ramulus, Bacteroides clostridiiformis ssp. clastridliformis,Bacteroides coagulans, Prevotella oralis, Prevotella ruminicola,Odoribacter splanchnicus, and Desuifomonas pigra.

In one aspect, a fecal microbe preparation lacks or is substantiallydevoid of one or more, one or more, two or more, three or more, four ormore, or five or more live fecal microorganisms are selected from thegroup consisting of Acidamiococcus, Akkermansia, Alistipes,Anaerotruncus, Bacteroides, Bifidobacterium, Blautia, Butyrivibrio,Clostridium, Collinsella, Coprococcus, Corynebacterium, Dorea,Enterococcus, Escherichia, Eubacterium, Faecalibacterium, Haemophilus,Holdemania, Lactobacillus, Moraxella, Parabacteroides, Prevotella,Propionibacterium, Raoultella, Roseburia, Ruminococcus, Staphylococcus,Streptococcus, Subdoligranulum, and Veillonella. In one aspect, a fecalmicrobe preparation lacks or is substantially devoid of one or more, oneor more, two or more, three or more, four or more, or five or live morefecal microorganisms are selected from the group consisting ofBacteroides fragilis ssp. vulgatus, Collinsella aerofaciens, Bacteroidesfragilis ssp. thetaiotaomicron, Peptostreptococcus productus II,Parabacteroides distasonis, Faecalibacterium prausnitzii, Coprococcuseutactus, Peptostreptococcus productus, Ruminococcus bromii,Bifidobacterium adolescentis, Gemmiger formicilis, Bifidobacteriumlongum, Eubacterium siraeum, Ruminococcus torques, Eubacterium rectale,Eubacterium eligens, Bacteroides eggerthii, Clostridium leptum.Bacteroides fragilis ssp. A, Eubacterium biforme, Bifidobacteriuminfantis, Eubacterium reciale, Coprococcus comes, Pseudoflavonifractorcapillosus, Ruminococcus albus, Dorea formicigenerans, Eubacteriumhallii, Eubacterium ventriosum, Fusobacterium russi, Ruminococcus obeum,Eubacterium rectale, Clostridium ramosum, Lactobacillus leichmannii,Ruminococcus callidus, Butyrivibrio crossotus, Acidaminococcusfermentans, Eubacterium entriosum, Bacteroides fragilis ssp. fragils,Coprococcus catus, Aerostipes hadrus, Eubacterium cylidroides,Eubacterium ruminantium, Staphylococcus epidermidis, Eubacteriumlimosum, Tissirella praeacuta, Fusobacterium mortiferum, Fusobacteriumnaviforme, Clostridium innocuum, Clostridium ramosum, Propionibacteriumacnes, Ruminococcus flavefaciens, Bacteroides fragilis ssp. ovatus,Fusobacterium nucleatun, Fusobacterium mortiferum, Escherichia coli,Genella morbillorum, Finegoldia magnus, Streptococcus intermedius,Ruminococcus lactaris, Eubacterium tenue, Eubacterium ramuls,Bacteroides clostridiiformis ssp. clostridliformis, Bacteroidescoagulans, Prevotella oralis, Prevotella ruminicola, Odoribactersplanchnicus, and Desuifomonas pigra.

In some aspects, a method of the present disclosure further comprisesallowing the subject to intake only water for up to two hours prior toorally administrating a single dose of the pharmaceutical composition.In certain aspects, a method of the present disclosure comprisesallowing the subject to intake only water for up to about half an hour,up to about one hour, up to about one and a half hour, or up to abouttwo hours prior to orally administrating a single dose of thepharmaceutical composition. In an aspect, the method of the presentdisclosure further comprises allowing the subject to intake only waterfor up to two hours after orally administrating a single dose of thepharmaceutical composition. In certain aspects, a method of the presentdisclosure comprises allowing the subject to intake only water for up toabout half an hour, up to about one hour, up to about one and a halfhour, or up to about two hours after orally administrating a single doseof the pharmaceutical composition. In some aspects, the method of thepresent disclosure requires no colon purgative prior to the oraladministering step. In certain aspects, the method of the presentdisclosure further comprises keeping the subject in an upright positionfor at least two hours after orally administrating a single dose of thepharmaceutical composition.

In some aspects, a method of the present disclosure further comprisesstoring the pharmaceutical composition at 4° C. or higher prior to theoral administering step. In an aspect, the method of the presentdisclosure further comprises storing the pharmaceutical composition atroom temperature for at least 3 days prior to the oral administeringstep.

In an aspect, the pharmaceutical composition used in methods of thepresent disclosure comprising a freeze-dried fecal microbe preparationmay comprise a cryoprotectant selected from the group consisting oftrehalose, glucose, fructose, sucrose, lactose, ribose, mannitol,erythritol, arabitol, sorbitol, alanine, glycine, proline, sand acombination thereof.

In some aspects, the pharmaceutical composition used in methods of thepresent disclosure may be formulated as an enteric coated capsule ormicrocapsule, an acid-resistant capsule, an acid-resistant microcapsule,an enteric coated tablet, an acid-resistant tablet, an enteric coatedgeltab, an acid-resistant geltab, an enteric coated pill, or anacid-resistant pill. In certain aspects, the pharmaceutical compositionof the present disclosure may be administered together with a food, aliquid beverage, a food additive, a dairy-based product, a soy-basedproduct or a derivative thereof, a jelly, or a yogurt.

In some aspects, a single dose in accordance with the present disclosurecomprises a total cell count of 10¹⁰ or lower, such as between about 10³and about 10¹⁰, between about 10⁴ and about 10¹⁰, between about 10⁵ andabout 10¹⁰, between about 10⁶ and about 10¹⁰, between about 10⁷ andabout 10⁹, or between about 10⁷ and about 10⁸. In certain aspects, asingle dose in accordance with the present disclosure comprises a totallive cell count of 10¹⁰ or lower, such as between about 10³ and about10¹⁰, between about 10⁴ and about 10¹⁰, between about 10⁵ and about10¹⁰, between about 10⁶ and about 10¹⁰, between about 10⁷ and about 10⁹,or between about 10⁷ and about 10⁸.

In one aspect, a single dose in accordance with the present disclosurecomprises at least about 10⁵, 10 ⁶, 10⁷, 10⁸, 10 ⁹, 10¹⁰, 10¹¹, 10¹², or10¹³ cfu. In another aspect, a single dose comprises at most about 10⁵,10⁶, 10 ⁷, 10⁸, 10⁹, 10 ¹⁰, 10¹¹, 10 ¹², or 10¹³ cfu. In a furtheraspect, a single dose is selected from the group consisting of from 10⁸cfu to 10¹⁴ cfu, from 10⁹ cfu to 10¹³ cfu, from 10¹⁰ cfu to 10¹² cfu,from 10⁹ cfu to 10¹⁴ cfu, from 10⁹ cfu to 10¹² cfu, from 10⁹ cfu to 10¹¹cfu, from 10⁹ cfu to 10¹⁰ cfu, from 10¹⁰ cfu to 10¹⁴ cfu, from 10¹⁰ cfuto 10¹³ cfu, from 10¹¹ cfu to 10¹⁴ cfu, from 10¹¹ cfu to 10¹³ cfu, from10¹² cfu to 10¹⁴ cfu, and from 10¹³ cfu to 10¹⁴ cfu. In one aspect, apharmaceutical composition comprises the foregoing single dose in a unitweight of about 0.2, 0.4, 0.6, 0.8 or 1.0 gram, or a unit volume ofabout 0.2, 0.4, 0.6, 0.8 or 1.0 milliliter.

In one aspect, a single dose in accordance with the present disclosurecomprises at least about 10⁵, 10 ⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², or10¹³ cells or spores. In another aspect, a single dose comprises at mostabout 10⁵, 10 ⁶, 10⁷, 10 ⁸, 10 ⁹, 10¹⁰, 10¹¹, 10¹², or 10¹³ total cellsor spores. In a further aspect, a single dose is selected from the groupconsisting of from 10⁸ to 10¹⁴, from 10⁹ to 10¹³, from 10¹⁰ to 10¹²,from 10⁹ to 10¹⁴, from 10⁹ to 10¹², from 10⁹ to 10¹¹, from 10⁹ to 10¹⁰,from 10¹⁰ to 10¹⁴, from 10¹⁰ to 10¹³, from 10¹¹ to 10¹⁴, from 10¹¹ to10¹³, from 10¹² to 10¹⁴, and from 10¹³ to 10¹⁴ cells or spores. In anaspect, the single dose cell count is directed to live cells. In oneaspect, a pharmaceutical composition comprises the foregoing single dosein a unit weight of about 0.2, 0.4, 0.6, 0.8 or 1.0 gram, or a unitvolume of about 0.2, 0.4, 0.6, 0.8 or 1.0 milliliter.

In some aspects, a single dose of pharmaceutical composition of thepresent disclosure in accordance with the present disclosure isadministered to a subject who had no prior exposure to fecalmicrobiota-based therapy. In certain aspects, a single dose ofpharmaceutical composition in accordance with the current disclosure mayeliminate or reduce gastrointestinal dysbiosis. In an aspect, a singledose of pharmaceutical composition in accordance with the currentdisclosure may increase bacterial diversity in a subject'sgastrointestinal tract.

In certain aspects, within 3 to 6 days from orally administering asingle dose of the pharmaceutical composition of the present applicationto a subject in need thereof, the relative abundance of Proteobacteriain the subject's stool may decrease by at least 30%. In certain aspects,the relative abundance of Proteobacteria in the subject's stool maydecrease by at least 30% within 3 to 5 days, within 3 to 4 days, orwithin 4 to 5 days from orally administering a single dose of thepharmaceutical composition of the present application. In some aspects,the relative abundance of Proteobacteria in the subject's stool maydecrease by at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 98%, within 3 to 6days from orally administering a single dose of the pharmaceuticalcomposition of the present application.

In an aspect, within 3 to 6 days from orally administering a single doseof the pharmaceutical composition of the present application to asubject in need thereof, the relative abundance of Firmicutes in thesubject's stool may increase by at least 30%. In certain aspects, therelative abundance of Firmicutes in the subject's stool may increase byat least 30% within 3 to 5 days, within 3 to 4 days, or within 4 to 5days from orally administering a single dose of the pharmaceuticalcomposition of the present application. In some aspects, the relativeabundance of Firmicutes in the subject's stool may increase by at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, or at least 98%, within 3 to 6 days from orallyadministering a single dose of the pharmaceutical composition of thepresent application.

In an aspect, within 3 to 6 days from orally administering a single doseof the pharmaceutical composition of the present application to asubject in need thereof, the relative abundance of Bacteroidetes in thesubject's stool may increase by at least 30%. In certain aspects, therelative abundance of Bacteroidetes in the subject's stool may increaseby at least 30% within 3 to 5 days, within 3 to 4 days, or within 4 to 5days from orally administering a single dose of the pharmaceuticalcomposition of the present application. In some aspects, the relativeabundance of Bacteroidetes in the subject's stool may increase by atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 98%, within 3 to 6 days from orallyadministering a single dose of the pharmaceutical composition of thepresent application.

In an aspect, within 3 to 6 days from orally administering a single doseof the pharmaceutical composition of the present application to asubject in need thereof, the alpha diversity of Firmicutes in thesubject's stool may increase by at least 20%. In certain aspects, thealpha diversity of Firmicutes in the subject's stool may increase by atleast 20% within 3 to 5 days, within 3 to 4 days, or within 4 to 5 daysfrom orally administering a single dose of the pharmaceuticalcomposition of the present application. In some aspects, the alphadiversity of Firmicutes in the subject's stool may increase by at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 70%, at least 80%, at least90%, at least 100%, at least 150%, or at least 200%, within 3 to 6 daysfrom orally administering a single dose of the pharmaceuticalcomposition of the present application.

In an aspect, within 6 days from orally administering a single dose ofthe pharmaceutical composition of the present application to a subjectin need thereof, the alpha diversity within Bacteroidetes in thesubject's stool remains substantially unchanged. In some aspects, thealpha diversity within Bacteroidetes in the subject's stool remainssubstantially unchanged within 21 days or 60 days from orallyadministering a single dose of the pharmaceutical composition of thepresent application. In certain aspects, the alpha diversity withinBacteroidetes in the subject's stool exhibits a change of less than 20%,such as less than 15%, less than 10%, less than 8%, less than 6%, orless than 4% within 6 days from orally administering a single dose ofthe pharmaceutical composition of the present application. In an aspect,the alpha diversity within Bacteroidetes in the subject's stool exhibitsa change of less than 20%, such as less than 15%, less than 10%, lessthan 8%, less than 6%, or less than 4% within 21 days from orallyadministering a single dose of the pharmaceutical composition of thepresent application. In another aspect, the alpha diversity withinBacteroidetes in the subject's stool exhibits a change of less than 20%,such as less than 15%, less than 10%, less than 8%, less than 6%, orless than 4% within 60 days from orally administering a single dose ofthe pharmaceutical composition of the present application.

In some aspects, a method in accordance with the present disclosure mayeliminate or reduces one or more, two or more, three or more, four ormore symptoms selected from the group consisting of diarrhoea, weightloss, bleeding, loss of appetite, abdominal pain, fever, and fatigue. Inan aspect, such elimination or reduction of symptom occurs within atleast 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, atleast 4 weeks, at least 5 weeks, at least 6 weeks from orallyadministering a single dose of the pharmaceutical composition of thepresent application.

In certain aspects, a method in accordance with the present disclosurefurther comprises providing a maintenance dosing schedule following theoral administration of a single dose. In some aspects, a maintenancedosing schedule comprises a dose lower or equal to the dose of thesingle dose. In an aspect, a maintenance dosing schedule lasts for aduration of at least about 2 months, at least about 4 months, at leastabout 6 months, at least about 8 months, at least about 10 months, atleast about 12 months, at least about 18 months, at least about 24months, at least about 36 months, at least about 48 months, at leastabout 72 months, or at least about 96 months. In certain aspects, theremay be an interval of at least 1 week between the single dose oraladministration and a maintenance dosing schedule. In some aspects, theinterval may be at least about 2 weeks, at least about 3 weeks, at leastabout 4 weeks, at least about 5 weeks, at least about 6 weeks, at leastabout 7 weeks, at least about 8 weeks, at least about 9 weeks, at leastabout 10 weeks, at least about 11 weeks, or at least about 12 weeks. Incertain aspects, the maintenance dosing schedule is a continuous dosingschedule. In an aspect, the maintenance dosing schedule is anintermittent dosing schedule. In some aspects, an intermittent dosingschedule comprises a treatment period of at least 1 days, at least 2days, at least 3 days, at least 4 days, at least 5 days, at least 6days, at least 7 days, at least 8 days, at least 9 days, at least 10days, at least 11 days, at least 12 days, at least 13 days, or at least14 days followed by a resting period of at least 1 days, at least 2days, at least 3 days, at least 4 days, at least 5 days, at least 6days, at least 7 days, at least 8 days, at least 9 days, at least 10days, at least 11 days, at least 12 days, at least 13 days, or at least14 days.

In certain aspects, a method in accordance with the present disclosurefurther comprises pretreating the subject with an antibiotic prior tooral administration of a single dose of the pharmaceutical compositionof the present disclosure. In some aspects, an antibiotic may beselected from the group consisting of amoxicillin, tetracycline,metronidazole, rifabutin, clarithromycin, clofazimine, vancomycin,rifampicin, nitroimidazole, chloramphenicol, and a combination thereof.In certain aspects, an antibiotic may be selected from the groupconsisting of rifaximin, rifamycin derivative, rifampicin, rifabutin,rifapentine, rifalazil, bicozamycin, aminoglycoside, gentamycin,neomycin, streptomycin, paromomycin, verdamicin, mutamicin, sisomicin,netilmicin, retymicin, kanamycin, aztreonam, aztreonam macrolide,clarithromycin, dirithromycin, roxithromycin, telithromycin,azithromycin, bismuth subsalicylate, vancomycin, streptomycin,fidaxomicin, amikacin, arbekacin, neomycin, netilmicin, paromomycin,rhodostreptomycin, tobramycin, apramycin, and a combination thereof.

In certain aspects, a method in accordance with the present disclosurefurther comprises pretreating the subject with an anti-inflammatory drugprior to oral administration of a single dose of the pharmaceuticalcomposition of the present disclosure.

In some aspects, a fecal microbe preparation of the present disclosuremay comprise a donor's entire or substantially complete microbiota. Incertain aspects, a fecal microbe preparation of the present disclosuremay comprise a non-selected fecal microbe. In some aspects, a fecalmicrobe preparation may comprise an isolated or purified population oflive non-pathogenic fecal bacteria from cultures. In certain aspects, afecal microbe preparation is substantially free of non-living matter. Insome aspects, a fecal microbe preparation is substantially free ofacellular material selected from the group consisting of residual fiber,DNA, viral coat material, and non-viable material. In an aspect, a fecalmicrobe preparation of the present disclosure may be substantially freeof eukaryotic cells from the donor of the fecal microbe. In someaspects, a fecal microbiota preparation of the present disclosurecomprises no antibiotic resistant population.

In certain aspects, a fecal microbe preparation of the presentdisclosure is prepared by a process comprising a treatment selected fromthe group consisting of ethanol treatment, detergent treatment, heattreatment, irradiation, and sonication, or a combination thereof. Insome aspects, a fecal microbe preparation of the present disclosure isprepared by a process not requiring one or more treatments selected fromthe group consisting of ethanol treatment, detergent treatment, heattreatment, irradiation, and sonication. In an aspect, a fecal microbepreparation of the present disclosure is prepared by a process withoutany one of the following treatments: ethanol treatment, detergenttreatment, heat treatment, irradiation, and sonication In one aspect, afecal microbe preparation of the present disclosure is prepared by aprocess involving a separation step selected from the group consistingof filtering, sieving, density gradients, filtration, chromatography,and a combination thereof. In one aspect, a fecal microbe preparation ofthe present disclosure is prepared by a process not requiring one ormore separation steps selected from the group consisting of filtering,sieving, density gradients, filtration, and chromatography.

In an aspect, a fecal microbe preparation of the present disclosure isprepared from reconstituted fecal material. In another aspect, a fecalmicrobe preparation of the present disclosure is prepared from syntheticfecal material.

In an aspect, a pharmaceutical composition provided or administeredherein comprises a fecal microbiota comprising a Shannon Diversity Indexof greater than or equal to 2.0, greater than or equal to 2.1, greaterthan or equal to 2.2, greater than or equal to 2.3, greater than orequal to 2.4, greater than or equal to 2.5, greater than or equal to3.0, greater than or equal to 3.1, greater than or equal to 3.2, greaterthan or equal to 3.3, greater than or equal to 3.4, greater than orequal to 3.5, greater than or equal to 3.6, greater than or equal to3.7, greater than or equal to 3.8, greater than or equal to 3.9, greaterthan or equal to 4.0, greater than or equal to 4.1, greater than orequal to 4.2, greater than or equal to 4.3, greater than or equal to4.4, greater than or equal to 4.5, or greater than or equal to 5.0. Inanother aspect, a pharmaceutical composition comprises fecal microbiotacomprising a Shannon Diversity Index of between 2.5 and 5.0, between 2.7and 5.0, between 2.9 and 5.0, between 3.1 and 5.0, between 3.3 and 5.0,between 3.5 and 5.0, between 3.7 and 5.0, between 3.9 and 5.0, orbetween 4.1 and 5.0. In one aspect, a Shannon Diversity Index iscalculated at the phylum level. In another aspect, a Shannon DiversityIndex is calculated at the family level. In one aspect, a ShannonDiversity Index is calculated at the genus level. In another aspect, aShannon Diversity Index is calculated at the species level. In a furtheraspect, a pharmaceutical composition comprises a preparation of flora inproportional content that resembles a normal healthy human fecal flora.

In a further aspect, a pharmaceutical composition comprises fecalbacteria from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 differentfamilies. In an aspect, a pharmaceutical composition provided oradministered herein comprises a fecal microbiota comprising no greaterthan 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%,2%, 3^(%), 4%, 5%, 6%, 7%, 8%, 9%, or 10% weight non-livingmaterial/weight biological material. In another aspect, a pharmaceuticalcomposition provided or administered herein comprises a fecal microbiotacomprising no greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95% weight non-living material/weightbiological material. In another aspect, a pharmaceutical compositionprovided or administered herein comprises, consists of, or consistsessentially of, particles of non-living material and/or particles ofbiological material of a fecal sample that passes through a sieve, acolumn, or a similar filtering device having a sieve, exclusion, orparticle filter size of 2.0 mm, 1.0 mm, 0.5 mm, 0.25 mm, 0.212 mm, 0.180mm, 0.150 mm, 0.125 mm, 0.106 mm, 0.090 mm, 0.075 mm, 0.063 mm, 0.053mm, 0.045 mm, 0.038 mm, 0.032 mm, 0.025 mm, 0.020 mm, 0.01 mm, or 0.2mm. “Non-living material” does not include an excipient, e.g., apharmaceutically inactive substance, such as a cryoprotectant, added toa processed fecal material. “Biological material” refers to the livingmaterial in fecal material, and includes microbes including prokaryoticcells, such as bacteria and archaea (e.g., living prokaryotic cells andspores that can sporulate to become living prokaryotic cells),eukaryotic cells such as protozoa and fungi, and viruses. In oneembodiment, “biological material” refers to the living material, e.g.,the microbes, eukaryotic cells, and viruses, which are present in thecolon of a normal healthy human. In an aspect, a pharmaceuticalcomposition provided or administered herein comprises an extract ofhuman feces where the composition is substantially odorless. In anaspect, a pharmaceutical composition provided or administered hereincomprises fecal material or a fecal floral preparation in a lyophilized,crude, semi-purified or purified formulation.

In an aspect, a fecal microbiota in a pharmaceutical compositioncomprises highly refined or purified fecal microflora, e.g.,substantially free of non-floral fecal material. In an aspect, a fecalmicrobiota can be further processed, e.g., to undergo microfiltrationbefore, after, or before and after sieving. In another aspect, a highlypurified fecal microbiota product is ultra-filtrated to remove largemolecules but retain the therapeutic microflora, e.g., bacteria.

In another aspect, a fecal microbiota in a pharmaceutical compositionused herein comprises or consists essentially of a substantiallyisolated or a purified fecal flora or entire (or substantially entire)microbiota that is (or comprises) an isolate of fecal flora that is atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.6%, 99.7%, 99.8% or 99.9% isolated or pure, or having no more thanabout 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 10% ormore non-fecal floral material; or, a substantially isolated, purified,or substantially entire microbiota as described in Sadowsky et al., WO2012/122478 A1, or as described in Borody et al., WO 2012/016287A2.

In an aspect, a fecal microbiota in a pharmaceutical compositioncomprises a donor's substantially entire or non-selective fecalmicrobiota, reconstituted fecal material, or synthetic fecal material.In another aspect, the fecal microbiota in a pharmaceutical compositioncomprises no antibiotic resistant population. In another aspect, apharmaceutical composition comprises a fecal microbiota and is largelyfree of extraneous matter (e.g., non-living matter including acellularmatter such as residual fiber, DNA, RNA, viral coat material, non-viablematerial; and living matter such as eukaryotic cells from the fecalmatter's donor).

In an aspect, a fecal microbiota in a pharmaceutical composition usedherein is derived from disease-screened fresh homologous feces orequivalent freeze-dried and reconstituted feces. In an aspect, a freshhomologous feces does not include an antibiotic resistant population. Inanother aspect, a fecal microbiota in a pharmaceutical composition isderived from a synthetic fecal composition. In an aspect, a syntheticfecal composition comprises a preparation of viable flora whichpreferably in proportional content, resembles normal healthy human fecalflora which does not include antibiotic resistant populations. Suitablemicroorganisms may be selected from the following: Bacteroides,Eubacterium, Fusobacterium, Propionibacteriuim, Lactobacillus,Ruminococcus, Escherichia coli, Gemmiger, Clostridium, Desldfomonas,Peptostreptococcus, Bifidobacterium, Coinsella, Coprococcus, Dorea, andRuminococcus.

In an aspect, a pharmaceutical composition is combined with otheradjuvants such as antacids to dampen bacterial inactivation in thestomach. (e.g., Mylanta, Mucaine, Gastrogel). In another aspect, acidsecretion in the stomach could also be pharmacologically suppressedusing H₂-antagonists or proton pump inhibitors. An example H-antagonistis ranitidine. An example proton pump inhibitor is omeprazole. In oneaspect, an acid suppressant is administered prior to administering, orin co-administration with, a pharmaceutical composition.

In some aspects, a fecal microbe preparation of the present disclosurecomprises a preparation of viable flora in proportional content thatresembles a normal healthy human fecal flora. In certain aspects, afecal microbe preparation of the present disclosure comprises bacteriafrom at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 12, at least 15,at least 18, or at least 20 different families. In some aspects, a fecalmicrobe preparation of the present disclosure comprises bacteria from atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 12, at least 15, at least 18,at least 20, at least 23, at least 25, at least 27, at least 30, atleast 32, at least 35, at least 38, or at least 40 different genera. Incertain aspects, a fecal microbe preparation of the present disclosurehas a Shannon Diversity Index of 0.4-5.0 at the family, genus, orspecies level.

In some aspects, a fecal microbe preparation of the present disclosurehas at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 99%, or at least about 99.5% microbes in a spore form. In certainaspects, a fecal microbe preparation of the present disclosure has atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 85%, at least about 90° %, at least about 95%, at leastabout 99%, or at least about 99.5% microbes in a non-spore form.

In an aspect, the present disclosure provides for the followingexemplary embodiments:

Embodiment 1: A method for treating a Clostridium difficile infection(CDI) in a subject in need thereof, said method comprising orallyadministering to said subject a single dose of a pharmaceuticalcomposition comprising a freeze-dried fecal microbiota preparation,wherein said single dose achieves a CDI clearance rate of at least 80%.

Embodiment 2: A method for treating a Clostridium difficile infection(CDI) in a subject in need thereof, said method comprising orallyadministering to said subject a single dose of a pharmaceuticalcomposition comprising a freeze-dried fecal microbiota preparation,wherein said single dose is capable of achieving a CDI clearance rate ofat least 80%.

Embodiment 3: The method of embodiment 1 or 2, wherein said CDIclearance rate is calculated based on a patient population size of 20,30, 40, 50, or 100.

Embodiment 4: The method of embodiment 1 or 2, wherein the relativeabundance of Proteobacteria in said subject's stool decreases by atleast 50% within 3 to 6 days from administering said single doserelative to a baseline abundance immediately prior to administering saidsingle dose.

Embodiment 5: The method of embodiment 1 or 2, wherein the relativeabundance of Firmicutes in said subject's stool increases by at least50% within 3 to 6 days from administering said single dose relative to abaseline abundance immediately prior to administering said single dose.

Embodiment 6: The method of embodiment 1 or 2, wherein the relativeabundance of Bacteroidetes in said subject's stool increases by at least50% within 3 to 6 days from administering said single dose relative to abaseline abundance immediately prior to administering said single dose.

Embodiment 7: The method of embodiment 1 or 2, wherein the alphadiversity within Firmicutes in said subject's stool increases by atleast 100% within 3 to 6 days from administering said single doserelative to a baseline diversity immediately prior to administering saidsingle dose.

Embodiment 8: The method of embodiment 1 or 2, wherein the alphadiversity within Bacteroidetes in said subject's stool remainssubstantially unchanged within 6 days, 21 days, or 60 days fromadministering said single dose relative to a baseline diversityimmediately prior to administering said single dose.

Embodiment 9: The method of embodiment 1 or 2, wherein the alphadiversity within Bacteroidetes in said subject's stool exhibits a changeof less than 15% within 6 days, 21 days, or 60 days from administeringsaid single dose relative to a baseline diversity immediately prior toadministering said single dose.

Embodiment 10: The method of embodiment 1 or 2, wherein said single doseachieves a CDI clearance rate of at least 85%, 88%, 90%, 92%, 94%, 96%,98%, or 99%.

Embodiment 11: The method of embodiment 1 or 2, wherein said subject isallowed only water for two hours prior to said administering saidpharmaceutical composition.

Embodiment 12: The method of embodiment 1 or 2, wherein said subject isallowed only water for two hours after said administering saidpharmaceutical composition.

Embodiment 13: The method of embodiment 1 or 2, wherein said subjectremains upright for at least two hours after said administering saidpharmaceutical composition.

Embodiment 14: The method of embodiment 1 or 2, wherein said subjectexperience little or no bowel movement irregularity, bloating, orflatulence within the first two weeks from administering said singledose.

Embodiment 15: The method of embodiment 1 or 2, wherein saidpharmaceutical composition is stored at 4° C. or higher prior to saidadministering.

Embodiment 16: The method of embodiment 1 or 2, wherein saidpharmaceutical composition is stored at 4° C. or lower prior to saidadministering.

Embodiment 17: The method of embodiment 1 or 2, wherein saidpharmaceutical composition is stored at −20° C. or −80° C. for long termstorage.

Embodiment 18: The method of embodiment 1 or 2, wherein saidpharmaceutical composition is capable being stored at room temperaturefor at least 3 days prior to said administering.

Embodiment 19: The method of embodiment 1 or 2, wherein said methodrequires no colon purgative prior to administering said single dose.

Embodiment 20: The method of embodiment 1 or 2, wherein saidpharmaceutical composition is formulated as an enteric coated capsule ormicrocapsule, an acid-resistant capsule or microcapsule, an entericcoated tablet, an acid-resistant tablet, an enteric coated geltab, anacid-resistant geltab, an enteric coated pill, or an acid-resistantpill.

Embodiment 21: The method of embodiment 1 or 2, wherein saidpharmaceutical composition is administered together with a food, aliquid beverage, a food additive, a dairy-based product, a soy-basedproduct or a derivative thereof, a jelly, or a yogurt.

Embodiment 22: The method of embodiment 1 or 2, wherein said single dosecomprises a total cell count of 10¹⁰ or greater.

Embodiment 23: The method of embodiment 1 or 2, wherein said single dosecomprises a total cell count of 10¹⁰ or lower.

Embodiment 24: The method of embodiment 1 or 2, wherein said single dosecomprises a total live cell count of 10¹⁰ or lower.

Embodiment 25: The method of embodiment 22, wherein said single dosecomprises a total cell count or a total live cell count between about10³; and about 10¹⁰, between about 10⁴ and about 10¹⁰, between about 10⁵and about 10¹⁰, between about 10⁶ and about 10¹⁰, between about 10⁷ andabout 10⁹, or between about 10⁷ and about 10⁸.

Embodiment 26: The method of embodiment 1 or 2, wherein saidfreeze-dried fecal microbiota preparation comprises a cryoprotectantselected from the group consisting of trehalose, glucose, fructose,sucrose, lactose, ribose, mannitol, erythritol, arabitol, sorbitol,alanine, glycine, proline, sand a combination thereof.

Embodiment 27: The method of embodiment 1 or 2, wherein said CDI isprimary CDI.

Embodiment 28: The method of embodiment 1 or 2, wherein said CDI isrecurrent CDI.

Embodiment 29: The method of embodiment 1 or 2, wherein said single doseis said subject's first ever fecal microbiota-based therapy.

Embodiment 30: The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation comprises a donor's entire or substantiallycomplete microbiota.

Embodiment 31: The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation comprises a non-selected fecal microbiota.

Embodiment 32: The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation comprises an isolated or purified population oflive non-pathogenic fecal bacteria from culturing.

Embodiment 33: The method of embodiment 1 or 2, wherein the preparationof said fecal microbiota preparation involves a treatment selected fromthe group consisting of ethanol treatment, detergent treatment, heattreatment, irradiation, and sonication, and a combination thereof.

Embodiment 34: The method of embodiment 1 or 2, wherein the preparationof said fecal microbiota preparation involves no treatment selected fromthe group consisting of ethanol treatment, detergent treatment, heattreatment, irradiation, and sonication.

Embodiment 35: The method of embodiment 1 or 2, wherein the preparationof said fecal microbiota preparation involves a separation step selectedfrom the group consisting of filtering, sieving, differentialcentrifugation, density gradient centrifugation, filtration,chromatography, and a combination thereof.

Embodiment 36: The method of embodiment 1 or 2, wherein the preparationof said fecal microbiota preparation does not require one or moreseparation steps selected from the group consisting of filtering,sieving, density gradients, filtration, and chromatography.

Embodiment 37: The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation is substantially free of non-living matter.

Embodiment 38: The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation is substantially free of acellular materialselected from the group consisting of residual fiber, DNA, viral coatmaterial, and non-viable material.

Embodiment 39: The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation is substantially free of eukaryotic cells fromsaid fecal microbe's donor.

Embodiment 40: The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation is from reconstituted fecal material.

Embodiment 41: The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation is from synthetic fecal material.

Embodiment 42. The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation comprises no antibiotic resistant population.

Embodiment 43: The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation comprises a preparation of viable flora inproportional content that resembles a normal healthy human fecal flora.

Embodiment 44: The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation comprises bacteria from at least 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 15, 18, or 20 different families.

Embodiment 45: The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation comprises bacteria from at least 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 15, 18, 20, 23, 25, 27, 30, 32, 35, 38, or 40 differentgenera.

Embodiment 46: The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation has a Shannon Diversity Index between 3.0 and 4.5at the species level.

Embodiment 47: The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation has at least about 20%, 30%, 40%, 50%, 60%, 70%,80%, 85%, 90%, 95%, 99%, or 99.5% microbes in a spore form.

Embodiment 48: The method of embodiment 1 or 2, wherein said fecalmicrobiota preparation has at least about 20%, 30%, 40%, 50%, 60%, 70%,80%, 85%, 90%, 95%, 99%, or 99.5% microbes in a non-spore form.

Embodiment 49: The method of embodiment 1 or 2, wherein said single doseis followed by a maintenance dosing schedule.

Embodiment 50: The method of embodiment 49, wherein said maintenancedosing schedule comprises a dose lower or equal to the dose of saidsingle dose.

Embodiment 51: The method of embodiment 49, wherein said second dosingschedule lasts for at least about 2, 4, 6, 8, 10, 12, 18, 24, 36, 48,72, or 96 months.

Embodiment 52: The method of embodiment 49, wherein the interval betweensaid single dose and said maintenance dosing schedule is at least about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

Embodiment 53: The method of embodiment 49, wherein said maintenancedosing schedule is a continuous dosing schedule.

Embodiment 54: The method of embodiment 49, wherein said maintenancedosing schedule is an intermittent dosing schedule.

Embodiment 55: The method of embodiment 54, wherein said intermittentdosing schedule comprises a treatment period of at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 days followed by a resting period ofat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

Embodiment 56: The method of any one of preceding embodiments, whereinsaid single dose eliminates or reduces gastrointestinal dysbiosis.

Embodiment 57: The method of any one of preceding embodiments, whereinsaid single dose increases bacterial diversity in said subject'sgastrointestinal tract.

Embodiment 58: The method of any one of preceding embodiments, whereinsaid subject is pretreated with an antibiotic prior to administration ofsaid composition.

Embodiment 59: The method of embodiment 58, wherein said antibiotic isselected from the group consisting of amoxicillin, tetracycline,metronidazole, rifabutin, clarithromycin, clofazimine, vancomycin,rifampicin, nitroimidazole, chloramphenicol, and a combination thereof.

Embodiment 60: The method of embodiment 58, wherein said antibiotic isselected from the group consisting of rifaximin, rifamycin derivative,rifampicin, rifabutin, rifapentine, rifalazil, bicozamycin,aminoglycoside, gentamycin, neomycin, streptomycin, paromomycin,verdamicin, mutamicin, sisomicin, netilmicin, retymicin, kanamycin,aztreonam, aztreonam macrolide, clarithromycin, dirithromycin,roxithromycin, telithromycin, azithromycin, bismuth subsalicylate,vancomycin, streptomycin, fidaxomicin, amikacin, arbekacin, neomycin,netilmicin, paromomycin, rhodostreptomycin, tobramycin, apramycin, and acombination thereof.

Embodiment 61: The method of anyone of preceding embodiments, whereinsaid subject is pretreated with an anti-inflammatory drug prior toadministration of said composition.

Embodiment 62: The method of any one of preceding embodiments, whereinsaid method eliminates or reduces one or more, two or more, three ormore, four or more symptoms selected from the group consisting ofdiarrhea, weight loss, bleeding, loss of appetite, abdominal pain,fever, and fatigue.

While the present disclosure has been described with reference topreferred embodiments, it will be understood by those skilled in the artthat various changes may be made and equivalents may be substituted forelements thereof to adapt to particular situations without departingfrom the scope of the present disclosure. Therefore, it is intended thatthe present disclosure not be limited to the particular embodimentsdisclosed as the best mode contemplated for carrying out the presentdisclosure, but that the present disclosure will include all embodimentsfalling within the scope and spirit of the appended claims.

EXAMPLES Example 1

Fecal bacteria are prepared using the standard methods as previouslydescribed in Hamilton et al. Am. J. Gastroenterology 2012; 107:761-7,except that glycerol is substituted with one of the followingcryoprotectants (all chemicals were USP grade or better and prepared inPBS, pH 7.0): 5% sucrose only; 10% sucrose only; 10% skim milk only; 5%trehalose only; 10% trehalose only; 10% trehalose plus 2.5% sucrose; 5%trehalose plus 2.5% sucrose; 5% mannitol only; or 10% mannitol only. Thelyophilizer (LyoStar II, Stone Ridge, N.Y., or equivalent) used has ashelf temperature of −20° C. for 36 hours followed by 6 hours at +30° C.All steps are done under 100 mT vacuum or less, and the final product isheld at +20° C. until used. The total dose is 2.5×10¹ cells.

Specifically, the cyroprotectants mannitol and trehalose, at 5% or 10%concentrations yield preparations that can be broken into a fine powderand easily packaged into capsules. However, viability of bacteria withtrehalose is superior compared to mannitol, as measured by theirmembrane integrity (Table 1) and nearly indistinguishable (˜4% loss ofviability) compared to fresh fecal microbiota. In particular, membraneintegrity is stable for up to 8 weeks post-lyophilization. Additionally,there is little difference in viability between preparations made with5% trehalose compared to 10% trehalose, with lesser amount of trehaloseallowing dosing in fewer capsules. Consequently, trehalose is chosen asthe standard cryoprotrectant in all further studies.

TABLE 1 Percent intact cell viability data from frozen (liquid) andlyophilized material obtained from a single sample at various timepointsafter processing/lyophilization. Raw material - 60.2% intact Time(weeks) Material after lyo 1 2 3 4 6 8 Frozen - 10% N/A 55.1 58.1 53.457.2 56.2 55.3 glycerol 10% Mannitol 31.7 34.4 33.4 32.2 36.3 29.2 34.25% Mannitol 29.5 32.1 27.6 30.1 28.6 31.3 28.4 10% Trehalose 56.7 57.455.3 55.6 53.1 56.4 52.1 5% Trehalose 59.4 55.4 58.9 57.2 56.2 52.2 55.8

Membrane integrity of freeze-dried microbiota remains intact after 96hours of storage at room temperature, 4° C. and −20° C. (Table 2). Cellcounts and membrane integrity are determined from triplicate samples offresh microbiota prepared following filtration steps and holding at roomtemperature, 4° C., and −20° C. for 96 hours. Results of this experimentshow that there is no significant difference in cell integrity insamples held at room temperature, 4° C., or −20° C., relative to thatfound in the initial preparation.

TABLE 2 Temperature stability of encapsulated freeze-dried microbiota.Counts/square Membrane 1 2 3 4 5 Average Cells/g Integrity SampleInitial 69 67 64 57 58 63 7.9E+11 49% 1 −20° C. 56 52 50 49 48 516.4E+11 51%  +4° C. 40 44 49 52 37 44 5.6E+11 58% Room Temperature 66 5657 55 49 57 7.1E+11 54% Sample Initial 57 55 53 60 57 56 7.1E+11 61% 2−20° C. 57 68 60 56 60 60 7.5E+11 63%  +4° C. 55 64 50 49 58 55 6.9E+1165% Room Temperature 67 62 74 56 55 63 7.9E+11 63% Sample Initial 89 8385 82 83 84 1.1E+12 55% 3 −20° C. 90 87 88 82 92 88 1.1E+12 68%  +4° C.72 68 73 66 70 70 8.7E+11 55% Room Temperature 95 78 81 91 83 86 1.1E+1249%

Example 2

Germ-free mice are bred and maintained in the germ free facility at theMayo Clinic (Rochester, Minn., USA). Animals are administered microbiotaor PBS via oral gavage, 100 μL per dose. Microbiota preparations includefrozen/thawed liquid with 10% glycerol, as described previously orrehydrated freeze-dried microbiota in 5% trehalose. The dosage to eachmouse, of either frozen or freeze-dried material, is 10¹⁰ cells. Fecalpellets are collected prior to gavage, as well as 3, 7, 14, and 21 daysfollowing gavage.

In order to ensure that the different taxa of microbiota are preservedby the freeze-drying protocol, preparations in germ free mice aretested. Comparison is made to frozen/thawed liquid preparation withglycerol. Prompt and stable engraftment of all bacterial phyla isevident for both frozen and freeze-dried treatment groups. FIG. 1A showsa distribution of phyla among all mouse fecal pellets and donor samples,without rarefication. FIG. 1B shows the Phylum-level classification ofOTUs that are associated with donor contribution. Error bars reflectstandard error of the mean.

Following gavage, engraftment proceeds as an early expansion of theBacteroidetes, predominantly among the families Porphyromonadaceae andBacteroidaceae, with a subsequent increase in the relative abundance ofFirmicutes, primarily the families Lachnospiraceae and Ruminococcaceae(FIG. 1A). Engraftment is evident for both frozen and freeze-driedtreatment groups at three days post-gavage (T₃; FIG. 1B), with donorOTUs accounting for >50% of the communities. The donor community appearsto establish more quickly using the freeze-dried preparation, butdifferences between preparation are not significant at T₃ (p=0.101) oracross all timepoints (p=0.237). Community composition of both frozenand freeze-dried treatment groups vary from each other as well as fromdonor communities by ANOSIM (p<0.001), and each group clustersindependently (AMOVA p<0.001, FIG. 2).

Example 3

Double-encapsulated capsules are prepared by using a filled size 0capsule packaged inside a size 00 capsule. Hypromellose capsules areDRcaps® from Capsugel (Morristown, N.J.). Capsules are manually filledusing a 24-hole filler (Capsule Machine, Capsule Connection, Prescott,Ariz.) to a final concentration of ˜1×10¹¹ cells/capsule. The capsulesare stored at −80° C. (a convenient dry storage option) in 50 mL conicaltubes until needed. Once taken out of the freezer, a dessicant packet isadded to the container. The length of storage period at −80° C. does notappear to impact the effectiveness of the capsules (Table 3).

TABLE 3 Storage duration of encapsulated microbiota prior to dispensingto the patients. Number of patients that underwent rescue capsulePatients that underwent FMT following failure of their first FMT usingDuration in colonoscopic FMT: capsule administration: −80° C. storagefailure/success failure/success 0-3 months 3/4 3/15 3-6 months 1/0 0/5 6-9 months 0/1 0/3  9-12 months  1/0 1/12

Example 4

All patients offered initial FMT in the University of Minnesota program,from its inception in 2008 inclusive of the experience described here,satisfy formal inclusion and exclusion criteria, described previously inKhoruts et al. Clin. Gastroenterol. Hepatol. 2016. Briefly, theseinclusion criteria are: (1) informed consent; (2) documentation of atleast two spontaneous relapses of CDI following the initial episode ofthe infection; (3) failure of at least one extended antibiotic regimen(≥6 weeks) to clear the infection; (4) documentation of CDI by stooltesting within three months of FMT. Exclusion criteria for all FMTpatients include: (1) anticipation of non-CDI antibiotic treatmentwithin three months of FMT; (2) life expectancy of less than two yearsif the patient is able to tolerate suppressive therapy with vancomycin,125 mg daily, or rifaximin for patients with liver disease and hepaticencephalopathy. In addition, exclusion criteria for FMT with anencapsulated oral preparation of FMT in this study include: (1)dysphagia, (2) known inflammatory bowel disease (IBD), (3) absence ofclinical indications for a diagnostic colonoscopy, (4) anyimmunosuppressive therapy or presence of known immune deficiency (e.g.,IgA deficiency), (5) failure to obtain informed consent for capsule FMT.Patients excluded from capsule FMT are offered colonoscopic FMT as anoption if the general inclusion/exclusion criteria are satisfied.

Some patients included in this cohort are recipients of previous FMT,administered via colonoscopy. In this program, patients suffering aspontaneous recurrence of CDI, i.e., relapse of the infection without anew antibiotic provocation, are offered another round of FMT. If thepatients suffer a re-infection with CDI, i.e., relapse following a newantibiotic provocation, they are offered one round of anti-CDIantibiotic (preferably fidaxomicin, metronidazole, or vancomycin) andFMT only following a spontaneous relapse following such antibiotictreatment attempt.

Donor material used in preparation of FMT capsules is obtained fromstandard donors as previously described (University of Minnesota IRBdonor protocol 1303M29782, Khoruts et al. Clin. Gastroenterol. Hepatol.2016). Material for capsule FMT preparations is provided by two maledonors. A medical personnel delivers the FMT capsules to patient homesand reinforces instructions for the FMT protocol. In the beginning, whenpatients are prepared with a colon purgative, vancomycin is continueduntil one day prior to FMT. Once the colon purgative is eliminated fromthe protocol, the patients discontinue vancomycin two days prior to FMT.The patients are told they could keep the capsules in the refrigeratorfor two days. The patients are allowed only water for two hours prior totaking the capsules. Only water is allowed for two hours after takingthe capsules during which time the patients had to remain upright. Theprotocol evolves in the course of clinical experience as describedfurther in the results section. Variables include (1) administration ofa colon purgative prior to capsule FMT (discontinued after the firstfour patients; (2) acid suppressive medications; (3) the dose ofFMT—this is decreased in the course of the study due to limited quantityof prepared material associated with reduction in key laboratorypersonnel.

In a single center (University of Minnesota) pragmatic study describingthe entire clinical experience with capsule FMT from June 2014 to March2016, failure of FMT is defined as spontaneous relapse of diarrhealsymptoms and positive stool testing for C. difficile toxin B by PCRwithin two months of administration. C. difficile toxin B is measured inall patients that noted or complained of loose stools, regardless offrequency. All patients are seen in clinic after two months for afollow-up clinic visit following capsule FMT and are instructed toremain in contact with the clinic indefinitely with any new questions orconcerns and any new prescriptions for antibiotics by other providers.The study is approved by the University of Minnesota InstitutionalReview Board.

The basic characteristics of the patients in this clinical cohort arecomparable to patients without underlying BD offered FMT in thisprogram. These patients fail all reasonable attempts at breaking thecycle of R-CDI with antibiotics alone.

The clinical capsule FMT protocol evolves over the course of theprogram. The initial dose (˜2.5×10¹² bacteria, 24-27 capsules) is basedon previous colonoscopic experience, which emerged from crude andarbitrary dosing based on stool weight. The capsules are administeredover 2-3 days, 2-3 times per day on an empty stomach. The first fourpatients are instructed to take a colon purgative, identical to the onethey would have received prior to colonoscopic FMT, before taking thecapsules. The fifth patient is a paraplegic for whom taking thepurgative presented an extreme difficulty. Therefore, she does notreceive the purgative, but instead lengthened the period off vancomycinto two days prior to initiating the capsule FMT. The clinical outcome issuccessful, and based on this anecdotal evidence the purgativepreparatory step is eliminated from the protocol for all other patientsonwards.

The patients may not immediately embrace the capsule FMT protocol,despite its relative ease of administration. During the consent process,extensive experience with the colonoscopic FMT and relative novelty ofthe new capsule protocol are communicated to patients. Patients,exhausted with the cycles of CDI recurrence, prefer a treatment withknown history of success. Therefore, the early patients electing thecapsule are predominantly ones who suffered either spontaneousrecurrence or antibiotic-triggered re-infection with C. difficile afterbeing treated with colonoscopic FMT. These patients welcome the optionwithout the colonoscopy, possibly because they feel discouraged and wantto try something new. In fact, over the first six months of capsule FMTavailability, four patients are treated with capsule FMT and 31colonoscopic FMTs are performed. However, the pace of acceptance of thecapsule FMT alternative gradually increases with the growth of clinicalexperience with this preparation and ability to inform the patientsabout the clinical outcomes.

Due to the limited supply of capsules, it is necessary to ration thedoses. At first, the initial dose is halved and the next 14 patientsreceive 1.25×10¹² bacteria (14 capsules taken within one day). Finally,the original dose is decreased by an order of magnitude and the last 30patients are dosed at 2.1-2.5×10¹¹ bacteria (2-4 capsules, singleingestion). The patients report no difficulty or esthetic concernstaking the capsules at any dose.

The success rate in clearing CDI is 83.8% (41/49 patients) in the entirecohort. The success rate among all patients for whom the capsuletreatment is their first FMT is 89.7% (35/39 patients). One of thesepatients receives broad-spectrum antibiotics during a hospitalizationfor a complex urinary infection (the patient had urinary stents) withina day following her capsule FMT and suffers a re-infection with CDI. Thesuccess rate in clearing CDI for the lowest dosage of microbiota is93.3% (28/30 patients) and 96.2% (25/26) for patients for whom thecapsule treatment is their first FMT. Two patients out of the entirecohort suffer a spontaneous relapse of CDI after the 2-month end-point.No serious adverse events is observed in this cohort with the exceptionof the mentioned patient with the urinary infection. Approximately athird of patients report some bowel movement irregularity, bloating, andflatulence in the initial weeks following capsule FMT.

Example 5

Fecal samples are collected by patients into sterile containers within aweek prior to FMT, on post-FMT days 3, 7, 14, and post-FMT months 1, 3,and 6-12. The samples are kept frozen until pick-up by the researchassistant and are transported on ice into the laboratory.

A 250-500 mg amount of human fecal material is extracted using thePowerSoil® DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad,Calif., USA) without deviation from the manufacturer's instructions. Themicrobiome is characterized from patients for whom capsule FMTrepresented their first intervention (i.e. no prior colonoscopic FMT)and those that experienced any recurrence of infection, regardless ofprior intervention. The V5+V6 hypervariable regions of the 16S rRNA geneare amplified at the University of Minnesota Genomics Center (UMGC,Minneapolis, Minn., USA) using the BSF7841064R primer set. (Sogin et al.Proc. Natl. Acad. Sci. USA 2006; 103:12115-20; Claesson et al. NucleicAcids Res 2010; 38:e200) Amplicons are gel purified and purifiedamplicons are pooled in equal amounts for sequencing. Paired-endsequencing is performed by UMGC at a read length of 300 nt using theIllumina MiSeq platform (Illumina, Inc., San Diego, Calif., USA).Sequencing data are recovered as fastq files and are deposited in theSequence Read Archive of the National Center for BiotechnologyInformation under BioProject accession numbers SRP071210 and SRP064361,for germ-free mice and donor/patient samples, respectively.

Sequence processing and analysis, unless otherwise noted, is performedusing MOTHUR ver. 1.34.0. (Schloss et al. Appl. Environ. Microbiol.2009; 75:7537-41, Staley et al. Jmicrobiol Methods 2015; 114:43-50).Fastq files for both forward and reverse reads are trimmed to 150 nt andpaired-end joined using fastq-join software. (Aronesty Open Bioinforma J201; 7:1-8). Sequences are quality trimmed at an average quality scoreof 35 over a window of 50 nt. Any sequence with homopolymers >8 nt, anambiguous base, or >2 mismatches from primer sequences is excluded.High-quality sequences are aligned against the SILVA database ver. 119(Pruesse E et al. Nucleic Acids Res 2007; 35:7188-96) and subjected to a2% pre-clustering step. (Huse et al. Environ Microbiol 2010;12:1889-98). Chimeras are identified and removed using UCHIME software.(Edgar et al. Bioinformatics 2011; 27:2194-200). For comparison betweensamples, the number of reads per sample is rarefied to 50,456 reads forgerm-free mice and 11,500 for patient comparisons. (Gihring et al.Environ Microbiol 2012; 14:285-90). Operational taxonomic units (OTUs)are assigned at 97% identity using the furthest-neighbor algorithm andtaxonomic assignments are made against the RDP14 database. (Cole et al.Nucleic Acids Res 2009; 37:D141-5). Donor community engraftment isdetermined as a percentage of recipient communities that could beattributed to donor samples using the default parameters of theSourceTracker software. (Knights et al. Nat. Methods 2011; 8:761-3)

In order to simplify the analysis, the characterization of the fecalmicrobiome is limited to samples obtained from patients for whom thecapsule treatment is their first FMT. All samples prior to FMT showmarkedly lower microbial diversity compared to the donor microbiota(Table 4). Similar to previous investigations of patients undergoing FMTfor R-CDI in this own program and others (van Nood et al. N. Engl. J.Med. 2013; 368:407-15; Hamilton et al. Gut Microbes 2013; 4:125-35;Weingarden et al Microbiome 2015; 3:10; Weingarden et al. Am J PhysiolGastrointest Liver Physiol 2014; 306:G310-9; Shankar et al. Microbiome2014; 2:13; Shahinas et al. Mbio 2012; 3; Seekatz et al. MBio 2014;5:e00893-14), these pre-FMT samples demonstrate markedly increasedrelative abundance of Proteobacteria (40.7±4.3% versus 2.0±0.5%,p<0.001) and a reduction in the relative abundances of Firmicutes(34.4±3.8% versus 55.4±3.3%, p=0.443) and Bacteroidetes (8.5±2 7% versus39.7±3 4%, p<0.001) compared to the donor samples. Capsule FMT isassociated with increased microbial diversity, contraction of relativeabundance of Proteobacteria, and an increase in the relative abundancesof Firmicutes and Bacteroidetes, discussed in detail below.Interestingly, the kinetics of these changes is not equivalent amongdifferent phyla. FIG. 3A shows the distribution of phyla in curedpatient and donor samples. Samples are collected prior to FMT (pre-FMT),within the first 6 days post-FMT (days), between 7 and 21 days post-FMT(weeks), between 30 and 60 days post-FMT (months), or after 2 monthspost-FMT (>2 months). FIG. 3B shows the phylum-level classification ofOTUs that are associated with donor contribution. Error bars reflectstandard error of the mean. In particular, the recovery of Bacteroidetesis somewhat delayed and its relative abundance do not stabilize untilafter one month following capsule FMT.

TABLE 4 Alpha diversity indices (mean ± SE) for microbial communities inpatient samples. Clinical Outcome Timepoint* N (individuals) n (sample)Shannon Donor Donor 3 5 3.95 ± 0.13 Cure pre-FMT 26 28 2.36 ± 0.17 Days25 25 3.40 ± 0.16 Weeks 27 34 3.63 ± 0.11 Months 11 14 3.48 ± 0.13 >2months 7 7 3.37 ± 0.13 Recurrence pre-FMT 8 10 2.97 ± 0.44 Days 8 9 3.36± 0.40 Weeks 8 11 3.39 ± 0.38 Months 4 4 3.27 ± 0.22 >2 months 2 2 2.94± 0.97 p-value <0.0001

In order to assess the contribution of donor microbiota engraftment tothe changes in the FMT-associated microbial community structure, theSourceTracker computer program is employed. Prior to FMT this analysisattributes a relatively low fraction (22.6±4.4%) of OTUs to the donorsin patient samples. Donor similarity increases at all time points aftercapsule FMT and is greatest at least one month following treatment.Analysis of fecal samples from patients that received high dose capsuleFMT (1.25-2.5×10¹² bacteria) versus low dose (2.1-2.5×10¹¹ bacteria) donot show any dose-dependent differences in microbial diversity changesor kinetics of engraftment, further discussed in supplementary results.Similarly, the microbiome analysis does not show any deleterious effectof proton pump inhibitors on capsule FMT). Finally, the fecal samplesfrom all patients that failed to clear CDI with one capsule FMT isanalyzed, as discussed further below. Following FMT, the microbiomes ofthese patients generally become taxonomically more similar to donors,except the one who receives antibiotics shortly following the treatment.However, the small number of patients precludes elucidation of a clearpattern predictive of FMT failure.

Among all donor and patient samples, a mean sample coverage of 99.1±0.1%is observed with a mean of 279±29 OTUs observed in each sample. Alphadiversity is significantly lower in pre-FMT patient samples than donorand post-FMT samples, regardless of clinical outcome, based on theShannon index (Table 4, p<0.0001). While the abundance of Firmicutesdoes not differ significantly following capsule FMT, alpha diversitywithin the Firmicutes is significantly lower in pre-FMT samples andthose from patients who experienced recurrence (FIG. 4, p<0.0001), butdifferences in alpha diversity within the Bacteroidetes, which does showsignificant differences in abundance, did not differ significantly(p=0.468)

Capsule dosage does not significantly affect alpha diversity as measuredby the Shannon index, with no significant differences between patientswho received 10¹¹ bacteria dosages (3.42±0.07) compared to 10¹² bacteria(3.72±0.18, p=0.187). Despite some variation in the relative abundancesof the predominant phyla (FIG. 5), capsule dosage also does notsignificantly affect relative abundances of phyla within singletimepoints (p>0.05). Similarly, the extent of engraftment, measured bySourceTracker, is not dose-dependent, with no differences in the percentof donor similarity at any timepoint (p≥0.920). Furthermore, followingthe two-month follow-up, patient communities and donor similarity arenearly identical regardless of dose. Similar to differences in dosage,Shannon diversity is not significantly affected by the use of protonpump inhibitors (PPI), with mean indices of 3.51±0.08 versus 3.50±0.10,for those on and off PPI, respectively (p=0.824). Use of PPI also doesnot significantly affect the relative abundances of major phyla (FIG. 6,p>0.05) or the extent of donor engraftment (p≥0.977) within a singletimepoint.

Patients who experienced recurrence show similar taxonomic compositionprior to FMT (FIG. 7), but the abundance of Proteobacteria in the daysfollowing FMT is observed to be greater than among patients who did notrelapse (p=0.013). While the microbiomes of several patients (i.e. P02,P03, and P05) return to donor-like assemblages primarily comprised ofFirmicutes and Bacteroidetes, communities also show decreases in alphadiversity within the first weeks following FMT, while other patientsmaintained a greater proportion of Proteobacteria. Notably, themicrobiome of the patient who is placed on antibiotics for UTI (P07) wasprimarily comprised of Proteobacteria. Thus, a clear trend in the shiftsin microbial community composition that might indicate failure of FMTrequire further investigation.

1. A method for treating a Clostridium difficile infection (CDT) in asubject in need thereof, said method comprising orally administering tosaid subject a single dose of a pharmaceutical composition comprising afreeze-dried fecal microbe preparation, wherein said single dose iscapable of achieving a CDT clearance rate of at least 80%.
 2. The methodof claim 1, wherein said CDI clearance rate is calculated based on apatient population size of 20, 30, 40, 50, or
 100. 3. The method ofclaim 1, wherein the relative abundance of Proteobacteria in saidsubject's stool decreases by at least 50% within 3 to 6 days fromadministering said single dose relative to a baseline abundanceimmediately prior to administering said single dose.
 4. The method ofclaim 1, wherein the relative abundance of one or more bacteria selectedfrom the group consisting of Firmicutes and Bacteroidetes in saidsubject's stool increases by at least 50% within 3 to 6 days fromadministering said single dose relative to a baseline abundanceimmediately prior to administering said single dose.
 5. The method ofclaim 1, wherein the alpha diversity within Firmicutes in said subject'sstool increases by at least 100% within 3 to 6 days from administeringsaid single dose relative to a baseline diversity immediately prior toadministering said single dose.
 6. The method of claim 1, wherein thealpha diversity within Bacteroidetes in said subject's stool remainssubstantially unchanged within 6 days, 21 days, or 60 days fromadministering said single dose relative to a baseline diversityimmediately prior to administering said single dose.
 7. The method ofclaim 1, wherein said single dose achieves a CDI clearance rate of atleast 90%.
 8. The method of claim 1, wherein said subject is allowedonly water for two hours prior to said administering said pharmaceuticalcomposition, is allowed only water for two hours after saidadministering said pharmaceutical composition, or both.
 9. The method ofclaim 1, wherein said subject experience little or no bowel movementirregularity, bloating, or flatulence within the first two weeks fromadministering said single dose.
 10. The method of claim 1, wherein saidpharmaceutical composition is capable of being stored at roomtemperature for at least 3 days prior to said administering.
 11. Themethod of claim 1, wherein said method requires no colon purgative priorto administering said single dose.
 12. The method of claim 1, whereinsaid pharmaceutical composition is formulated as an enteric coatedcapsule or microcapsule, an acid-resistant capsule or microcapsule, anenteric coated tablet, an acid-resistant tablet, an enteric coatedgeltab, an acid-resistant geltab, an enteric coated pill, or anacid-resistant pill.
 13. The method of claim 1, wherein said single dosecomprises a total cell count of 10¹⁰ or greater.
 14. The method of claim1, wherein said freeze-dried fecal microbe preparation comprises acryoprotectant selected from the group consisting of trehalose, glucose,fructose, sucrose, lactose, ribose, mannitol, erythritol, arabitol,sorbitol, alanine, glycine, proline, sand a combination thereof.
 15. Themethod of claim 1, wherein said CDI is primary CDI or recurrent CDI. 16.The method of claim 1, wherein said fecal microbe preparation comprisesa donor's entire or substantially complete microbiota.
 17. The method ofclaim 1, wherein the preparation of said fecal microbe preparationinvolves a separation step selected from the group consisting offiltering, sieving, differential centrifugation, density gradientcentrifugation, filtration, chromatography, and a combination thereof.18. The method of claim 1, wherein said fecal microbe preparationcomprises a preparation of viable flora in proportional content thatresembles a normal healthy human fecal flora.
 19. The method of claim 1,wherein said subject is pretreated with an antibiotic prior toadministration of said composition.
 20. The method of claim 19, whereinsaid antibiotic is selected from the group consisting of amoxicillin,tetracycline, metronidazole, rifabutin, clarithromycin, clofazimine,vancomycin, rifampicin, nitroimidazole, chloramphenicol, and acombination thereof.